Carboxyl-modified single-walled carbon nanotubes negatively affect bacterial growth and denitrification activity

Abstract

Single-walled carbon nanotubes (SWNTs) have been used in a wide range of fields, and the surface modification via carboxyl functionalization can further improve their physicochemical properties. However, whether carboxyl-modified SWNT poses potential risks to microbial denitrification after its release into the environment remains unknown. Here we present the possible effects of carboxyl-modified SWNT on the growth and denitrification activity of Paracoccus denitrificans (a model denitrifying bacterium). It was found that carboxyl-modified SWNT were present both outside and inside the bacteria, and thus induced bacterial growth inhibition at the concentrations of 10 and 50 mg/L. After 24 h of exposure, the final nitrate concentration in the presence of 50 mg/L carboxyl-modified SWNT was 21-fold higher than that in its absence, indicating that nitrate reduction was substantially suppressed by carboxyl-modified SWNT. The transcriptional profiling revealed that carboxyl-modified SWNT led to the transcriptional activation of the genes encoding ribonucleotide reductase in response to DNA damage and also decreased the gene expressions involved in glucose metabolism and energy production, which was an important reason for bacterial growth inhibition. Moreover, carboxyl-modified SWNT caused the significant down-regulation and lower activity of nitrate reductase, which was consistent with the decreased efficiency of nitrate reduction.

Figure 1. SEM images of P. denitrificans cells in the absence (A) and presence of 50 mg/L non-modified SWNT (B) and carboxyl-modified SWNT (C) after 24 h of exposure.

Figure 2. Growth curves of P. denitrificans in the absence (control) and presence of non-modified SWNT and carboxyl-modified SWNT during 24 h of exposure.

Error bars represent standard deviations of triplicate measurements.

Figure 3. Transformations of NO₃⁻-N (A, solid line), NO₂⁻-N (A, dashed line) (A) and N₂O in the gas (B, solid line) and liquid phases (B, dashed line) in the absence (control) and presence of non-modified SWNT and carboxyl-modified SWNT.

Error bars represent standard deviations of triplicate measurements.

Figure 4. GO analysis of the sequencing reads from the exposure experiments with (A) and without (B) the presence of 50 mg/L carboxyl-modified SWNT.

Figure 5. Distribution of differentially expressed genes (red dots) caused by 50 mg/L carboxyl-modified SWNT in MA plot (A) and volcano plot (B).

Red and black dots represent the genes with significantly differential expression (>2-fold change and FDR < 0.05) and with no significant difference, respectively.

Figure 6. Gene expressions of key DNA sequences in the absence (control) and presence of 50 mg/L carboxyl-modified SWNT.

The gene expression level was calculated using the RPKM (reads per kilobase of exon region per million mapped reads) method.

Figure 7. Gene expressions (A) and relative activities (B) of nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (N₂OR) in the absence (control) and presence of 50 mg/L carboxyl-modified SWNT.

Error bars represent standard deviations of triplicate measurements. Asterisks indicate statistical differences (p < 0.05) from the control test.

Figure 8. TEM images of P. denitrificans cells in the absence (A) and presence of 50 mg/L carboxyl-modified SWNT (B) after 24 h of exposure.